In Staphylococcus aureus, an important human pathogen, the SaeRS two component system (TCS) is essential for producing multiple virulence factors and enhancing the bacterium's survival in the host. In this application, a novel approach will be explored to develop anti-staphylococcal drugs by targeting the SaeRS TCS. Although conventional antibiotics directly kill bacteria and suffer from resistance problems, the new anti-staphylococcal agent will reduce only the pathogenic potential of the bacterium without killing; therefore, the agent is expected to complement the conventional antibiotics regimen without a resistance problem. In the R21 phase, we will identify potent two lead candidates of the Sae-inhibitor. In Aim 1, promising hit compounds will be identified by screening 80,000 small molecules. The identified hit compounds will be further examined for their toxicity on a human cell line. In Aim 2, through pharmacokinetic tests, the hit compounds with good metabolic stability and low in vivo toxicity will be selected. In Aim 3, the selected compounds will be furthr examined for their in vivo efficacy in a murine model of infection; and two lead candidates will be identified for the R33 phase study. Only when the two lead candidates satisfy the R21 milestones, will the R33 phase be undertaken. In the R33 phase, the two lead candidates will be further improved, while their potential as a novel anti- staphylococcal drug will be thoroughly examined. In Aim 1, derivatives of the lead candidates will be generated; then the derivatives with improved pharmacological traits will be identified through efficacy and stability tests. In Ai 2, the lead candidates will be examined for their synergy with conventional antibiotics in treating staphylococcal infections. In Aim 3, the mechanism by which the lead candidates inhibit the SaeRS TCS will be determined by identifying the direct binding targets of the lead candidates and isolating resistant mutants. Finally, in Aim 4, the lead candidates will be thoroughly examined for their potential to cause emergence of resistant mutants in a condition similar to clinical settings. The overall goal of this study is to determine whether the inhibitors of the SaeRS TCS can be a novel anti-staphylococcal drug in combating the antibiotic resistance problem of S. aureus.